1. Field of the Invention
The present invention relates generally to clotting time testing, such as prothrombin time (PT) testing and, more specifically, to disposable clotting time testing devices and methods for assaying the clotting time of biological fluids without the aid of an instrument.
2. Description of the Background Art
Keeping blood in a fluid state, termed hemostasis, requires a subtle balance of pro- and anti-coagulants. Procoagulants prevent excessive bleeding by blocking blood flow from a damaged vessel, whereas anticoagulants prevent clots from forming in the circulation which could otherwise block blood vessels and lead to myocardial infarction.
The biochemical sequence leading to a blood clot is termed the coagulation cascade. The mechanism is based on catalytic conversion of fibrinogen, a soluble plasma protein, to insoluble fibrin. The enzyme catalyzing this reaction is thrombin, which does not permanently circulate in the blood in an active form, but exists as prothrombin, the inactive precursor of thrombin. Conversion to thrombin occurs in the presence of calcium ions and tissue thromboplastin. This mechanism is known as the extrinsic pathway. A second, more complex, intrinsic pathway is activated by clotting factors associated with platelets.
There are an estimated two million patients with cardiovascular disease in the United States who are currently on anticoagulation therapy. Thirty percent of these patients are xe2x80x9chigh riskxe2x80x9d for subsequent complications. Complications, when they occur, account for 400,000 hospitalizations and 200,000 deaths annually in the United States. Therefore, it is important to monitor these patients frequently to help prevent hemorrhagic and thromboembolic complications. Monitoring is accomplished by measuring the patient""s blood clotting time, more specifically, prothrombin time, usually weekly. During initial adjustment of anticoagulation medication, testing may occur more frequently than weekly.
Diagnosis of hemorrhagic conditions such as hemophilia, where one or more of the twelve blood clotting factors may be defective, can be made using a coagulation test. In addition, several tests have been developed to monitor the progress of thrombolytic therapy. Other test have been developed to signal a prethrombolytic or hypercoagulable state, or monitor the effect of administering protamine to patients during cardiopulmonary bypass surgery.
Currently, all prothrombin time testing is conducted on some form of instrumentation. These instruments range from desktop units used in clinical laboratories to small portable units designed to be used for point of care applications. The convenience of self-testing at home is the most effective means for driving patient compliance. Although portable instruments designed for home use are available, the high initial cost, the complexity of quality control, and the inconvenience of refrigerated storage of the reagent has not created a wide acceptance of these instruments by the patients. Thus, there exists a need for a non-instrumented clotting time test device that is simple to use, light in weight, inexpensive, and accurate.
The prothrombin test was first described by A. J. Quick, Am. J. Med. Sci 190:501 (1935), and involved mixing tissue thromboplastin with blood, under controlled conditions, to initiate coagulation via the extrinsic pathway. The standard laboratory technique for coagulation testing usually uses a turbidimetric method, such as described in U.S. Pat. No. 4,497,774. In this test, citrated plasma is mixed with tissue thromboplastin at 37xc2x0 C., and measurement is based upon optical density. An alternative method using calorimetric measurement is also described by Beker et al. in Hemostatis 12:73, 1982.
Moyer et al., in U.S. Pat. No. 3,951,606, first disclosed a manually operable disposable device which has a uniform bore reaction tube and limiting orifice for prothrombin time testing. A blood sample is applied to the device and mixed with activator reagent. The gravity-assisted flow travels through the reaction tube and halts when the blood clots. The distance traveled by the sample is proportional to the prothrombin time. Unfortunately, this device is difficult to fabricate and the end point generated is unstable because the flow continues slowly even after clotting has occurred.
U.S. Pat. No. 5,628,961, discloses an apparatus for conducting a variety of assays that are responsive to a change in the viscosity of a fluid sample. A reversible pump is used to move the fluid sample, making this device rather cumbersome.
Point of care whole blood prothrombin time instruments are described in U.S. Pat. Nos. 4,756,884 and 4,963,498. These instruments include cassettes with capillary channels in which the instrument monitors the change of flow of clotting blood. U.S. Pat. No. 5,004,923, describes additional optical and mechanical features of the instrument and cartridge that perform prothrombin time assays.
Other methods for measuring blood clotting include an instrument that measures the change of the field of magnetic particles inside disposable channels (U.S. Pat. No. 5,350,676); an instrument with miniature pumps and disposable cassettes with narrow restriction that plug up upon the clotting of blood (U.S. Pat. Nos. 5,591,403 and 5,731,212); a solid phase membrane with a dry reagent that contains a substrate which produces a detectable signal upon activation by a component of the coagulation pathway (U.S. Pat. No. 5,418,141); an instrument that uses a liquid crystal to measure clotting (U.S. Pat. No. 5,908,786); and piezoelectric means to detect clotting (U.S. Pat. No. 6,200,532).
U.S. Pat. No. 5,087,556, describes a non-instrumented measuring device which uses a flow channel with a detection film inside. This device, however, is not for use in conducting a clotting test. U.S. Pat. No. 4,756,884, teaches the use of a capillary channel containing reagent for estimating the flow change of blood. Although the surface energy inside the capillary channel, was defined, there is no description of how to control the flow stoppage of the blood upon clotting.
U.S. Pat. No. 6,113,855, describes a capillary flow channel including capillary-inducing structures, hexagonal and other geometric shapes, in which one end of the flow channel is deeper than the other end. There is no indication that this device can be used for clotting assays. Exner, in U.S. Pat. No. 5,601,995, discloses a method for estimating coagulation time by measuring the extent and rate of spreading of a blood sample deposited onto a porous sheet. Zweig et al., in U.S. Pat. No. 5,418,141, disclose test articles suitable for prothrombin time testing that comprise a solid phase membrane having dry thromboplastic immobilized thereon or within. A blood sample is applied to the application side, and a fluorescent signal is measured on the other side of the membrane.
Oberhardt et al. disclose another format in Y U.S. Pat. Nos. 5,110,727 and 5,350,676, based upon the use of magnetic particles mixed into a dry reagent contained within a flat capillary chamber. An applied oscillating magnetic field causes the particles to oscillate once the reagent is dissolved in blood. When the blood clots, the motion is diminished and the prothrombin time is estimated. Davis et al., in U.S. Pat. No. 5,628,961, disclose a method and device that respond to changes in the viscosity of a fluid sample using a reciprocating pump.
Thus, there remains a need for an inexpensive, non-instrumented disposable device for quantitative determination of prothrombin time that is both accurate and simple to use.
It is an object of the present invention to overcome the aforesaid deficiencies in the background art.
It is another object of the present invention to provide a self-contained quantitative analytical device that detects the end point of a clotting and agglutinating process.
It is another object of the present invention to provide a method for quantifying a clotting or agglutinating event.
The present invention relates to an analytical device for detecting a clotting or agglutinating event in a clottable or agglutinatable biological fluid comprising:
a. a capillary flow channel means through which the fluid may flow and in which the clotting or agglutinating event can occur;
b. A reagent to react with the clottable or agglutinatable biological fluid to produce a clot; and
c. A control means for controlling the flow rate of the clottable biological fluid and the usual appearance of the clot.
In a preferred embodiment, the device of the present invention contains a sample accepting area, a dried thromboplastic activator which optionally is impregnated on a reagent pad, a capillary channel that has a hydrophilic inner surface for drawing the sample along the channel and a hydrophilic woven fabric surface that controls the flow, provides wicking action, and allows the clotted or agglutinated mixture to anchor onto the fabric surface and stop flow.
The present invention also provides a method for quantifying the time for a clotting or agglutinating event using the device of the present invention. A sample fluid is received in the sample accepting area, and the sample fluid is mixed with reagent. The mixture is drawn through the capillary channel by capillary and/or wicking action. The flow of the sample mixture through the flow channel stops when the clotting or agglutinating event occurs.
For purposes of the present invention, xe2x80x9cclotting eventxe2x80x9d and xe2x80x9cagglutinating eventxe2x80x9d mean the endpoints of clot formation or agglutination, and can be used interchangeably.